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Abstract

Background

A couple of studies indicate a favorable impact of lupin protein on cardiovascular
risk factors in humans. These studies, however, used relatively high doses of > 33 g/d,
which can hardly be consumed under physiological conditions. Therefore, we investigated
the effect of 25 g/d lupin protein isolate (LPI) on selected cardiovascular markers
and on serum amino acids.

Methods

A total of 33 hypercholesterolemic subjects participated in a randomized, controlled,
double-blind crossover study. LPI and the active comparator milk protein isolate (MPI)
were incorporated in protein drinks and consumed over 8 wk separated by a 4 wk washout
period. Anthropometric data, blood pressure, and nutrient intake were assessed at
baseline and after 8 wk of both protein interventions. Blood was sampled at baseline,
wk 4 and wk 8. All 33 subjects were included in final statistical analyses using repeated
measures ANOVA with the general linear model or using linear mixed model.

Results

Except for higher HDL cholesterol at wk 4 of LPI (P ≤ 0.036), anthropometric parameters, blood pressure, and plasma lipids did not differ
among LPI and MPI intervention. Compared to baseline, the primary outcome LDL cholesterol
was significantly reduced after 4 wk of both interventions (P ≤ 0.008), while LDL:HDL cholesterol ratio was decreased only by LPI (P = 0.003). These time effects were restricted to subjects with higher hypercholesterolemia
and disappeared after 8 wk. Blood pressure was reduced after 8 wk of LPI (P ≤ 0.044). Almost all serum amino acids were higher at wk 4 but not at wk 8 of MPI
compared to LPI. Following 4 wk and 8 wk of LPI intervention, most amino acids remained
unchanged. Both interventions caused a slight, but significant rise in body weight
and body fat after 8 wk (P ≤ 0.045).

Conclusion

In conclusion, 25 g LPI can beneficially modulate plasma LDL cholesterol at least
over short-term. Using appropriate dietetic conditions that improve consumer compliance
and avoid changes in energy intake as well as in body composition, lupin protein could
positively impact cardiovascular risk factors particularly in individuals with higher
hypercholesterolemia.

Trial registration

Keywords:

Background

Replacing of animal by plant protein in foods is currently an important topic of discussion
due to the ecological and physiological benefits associated with vegetable sources
of proteins. In view of the growing global population as well as the limited availability
of agricultural land, there is an urgent need for high quality proteins from sustainable
plant sources such as legumes (e.g., soy, pea, and lupin). Moreover, the rising incidence of cardiovascular diseases
increases the demand for potential dietary interventions that could reduce the related
risk factors. For established cardiovascular conditions such as coronary heart diseases
and also for subjects at high risk, drug therapy is the recommended form for reducing
elevated LDL cholesterol concentrations [1]. However, ancillary to an existing therapy or for the primary prevention of coronary
heart diseases, non-pharmacological strategies such as weight reduction, increased
physical activity, and healthier dietary habits are endorsed by the National Cholesterol
Education Program (NCEP) [1].

Dietary proteins from plant sources can exert nutraceutical activities such as reduce
blood lipids and lower blood pressure [2-5], and thus constitute a healthier diet. As reviewed by Sirtori et al. [2], investigations in animals have revealed that proteins derived from either white
lupin (Lupinus albus) or blue lupin (Lupinus angustifolius) improve the lipoprotein profile and lower blood pressure. Most of the studies in
humans evaluated the physiological effects of lupin flour or lupin fiber, and only
a small number of investigations focused on the effects of lupin protein [6]. These studies observed a beneficial influence of lupin protein on blood cholesterol
concentrations [7-9] and also partially on blood pressure [7]. Furthermore, the study by Naruszewicz et al. [7] revealed a significant reduction of the inflammatory marker “high-sensitivity C-reactive
protein” (hs-CRP) after 90 d of lupin protein intake in hypercholesterolemic subjects.
As shown in a prospective study in women, hs-CRP is a strong predictor of the risk
of cardiovascular events [10]. The effect of lupin protein on the distribution of serum amino acids has only been
examined in one human trial [8]. Most importantly, in all of these studies, relatively high doses comprising more
than 30 g/d lupin protein were administered [7-9]. Such high daily doses can hardly be consumed under normal physiological conditions.
For example, for soy protein, which is closely related to lupin protein, the US Food
and Drug Administration established a health claim in 1999 stating that the intake
of 25 g/d soy protein beneficially affects serum lipids in humans [11]. Thus, further studies are needed to firstly, evaluate the impact of an equivalent
modest amount of lupin protein on cardiovascular health and secondly, to clarify the
effect on serum amino acids.

Methods

Subjects

A total of 65 volunteers aged between 18 and 80 years were recruited in the region
of Jena. Eligibility criterion was a total cholesterol concentration of ≥ 5.2 mmol/L
at screening, determined either by a general practitioner or on-site using a hand-held
point of care device from capillary blood (Accutrend® Plus System, Roche Diagnostics,
Grenzach-Wyhlen, Germany). Exclusion criteria were treatment with lipid-lowering drugs,
intake of nutritional supplements, which potentially influence lipid metabolism, and
intolerance, allergy or a strong dislike to any food ingredient present in the protein
drinks used in the study. In addition, breast-feeding mothers or pregnant females
were excluded. Thus, 33 eligible participants (18 females, 15 males) were invited
to an in-person meeting. Here, participants were offered essential study-relevant
information and also provided with a study folder containing print information. Written
informed consent was obtained from all subjects before start of the study. The study
protocol was approved by the Ethics Committee of the Medical Faculty of the Friedrich
Schiller University, Jena (no.: 2607-07/09).

Study design

The current study was part of a larger investigation consisting of two intervention
studies examining the influence of two different daily doses of LPI: 25 g (present
study) and 40 g [12] and comparing these with the effects of the respective doses of MPI. The present
study used a randomized, double-blind crossover design consisting of two 8 wk intervention
periods separated by a 4 wk washout period. The study was conducted between March
and August 2011 at the Department of Nutritional Physiology, Friedrich Schiller University
of Jena. Before commencement subjects were randomly assigned to one of two randomization
groups using computer-generated random numbers. One group received LPI to start with
(group AB) and the other group MPI first (group BA, Figure 1). Research assistants involved in the randomization procedure did not have access
to any information regarding demographic or laboratory characteristics of the subjects.
Moreover, protein drinks were labeled with numeric codes and all research assistants
as well as the participants were blinded to group assignments.

Study products

The daily portion of 25 g LPI or MPI was dissolved in 500 mL water. The protein drinks
produced by Nutrichem diät + pharma GmbH (Roth, Germany) were assembled as a 4 wk
supply in 250 mL sealed packages. Thus, 100 mL of the protein drink contained either
5.0 g LPI or the iso-nitrogenous amount of 5.1 g MPI. The amino acid compositions of the protein drinks
are shown in Table 1. Fat (3.0 g/100 mL) and carbohydrates (3.5 g/100 mL) were added to obtain a pleasant
taste and texture and to mask potential differences between the protein drinks. Subjects
were instructed to maintain their usual level of physical activity as well as their
dietary habits throughout the whole study. However, subjects were advised to replace
an iso-caloric part of their usual diet with the protein drinks since these provided an
additional energy intake of 1190 kJ/d.

Table 1.Relative amino acid composition of the two protein drinks administered in the study

The LPI was provided by the Fraunhofer Institute for Process Engineering and Packaging
(Fh-IVV, Freising, Germany) in the form of the protein isolate type E. It was produced
from the seeds of Lupinus angustifolius cv. Boregine as described by D'Agostina et al. [13]. This LPI contained 81.6 ± 1.3% protein (nitrogen × 5.8) and low quantities of water
(5.7 ± 0.0%), ash (4.4 ± 0.0%), fiber (6.0 ± 0.4%), and fat (1.4 ± 0.1%) in fresh
matter. In general, LPI type E contains the conglutins α, β, and δ [14] and is almost free of conglutin γ. Furthermore, it has low quantities of alkaloids
represented only by lupanine (34.1 ± 3.1 mg/kg) [15].

Data collection

At baseline participants recorded their usual daily eating patterns in a 5 d food
record with a precise documentation of weights and types of all consumed foods and
beverages. The composition of this basal diet was estimated with the use of PRODI®
5.9 (Nutri-Science GmbH, Freiburg, Germany). At the end of each intervention period,
subjects consumed a standard diet including the protein drinks. This standard diet
was prepared and preweighed in the study center and contained all foods required per
subject over 2 d. Subjects were instructed to consume no other foods, except for water.
Food intake was calculated by weighing food residues.

Body weight, body composition as well as blood pressure were determined at baseline
and after 8 wk of each intervention period. Fasting participants were weighed with
light clothes and without shoes using a digital scale. Body composition was determined
using bioelectrical impedance analysis (BIA 2000-S, Data Input GmbH, Darmstadt, Germany).
Blood pressure was measured in a sitting position in duplicate after 10 min of rest
on the left arm using an automatic blood pressure monitor (boso-medicus uno, Bosch + Sohn
GmbH u. Co. KG, Jungingen, Germany).

Blood samples were collected at baseline, and after 4 wk and 8 wk of each intervention
period. Following 12 h overnight fasting, blood samples were drawn by venipuncture
into a serum gel tube and a plasma gel tube containing lithium heparin (Sarstedt AG
& Co., Nümbrecht, Germany). Serum tubes were centrifuged at 20°C, 2500 × g for 10 min, the serum supernatants were aliquoted and stored at –80°C until analysis.
Plasma gel tubes were centrifuged at 15°C, 4302 × g for 7 min.

Analytical methods

Fresh plasma was analyzed for total, LDL, and HDL cholesterol as well as for triacylglyceroles,
urea, and hs-CRP according to the protocols of the Institute of Clinical Chemistry
and Laboratory Medicine, Jena University Hospital and quantified using the autoanalyzer
ARCHITECT C16000 (Abbott, Illinois, USA). For the analysis of free amino acids in
serum, the method based on the European Community Directive [17] was applied as described previously [18].

Statistical analyses

Statistical analyses were conducted using PASS 6.0 (NCSS Statistical Software, Kaysville,
UT, USA) or SPSS 19.0 (SPSS Inc., Chicago, USA). In all analyses, differences were
considered as statistically significant with P ≤ 0.050. A power analysis revealed > 80% power for the present study to detect a
10% difference in the primary outcome measure LDL cholesterol. All collected data
were tested for normal distribution and for homogeneity of variances applying the
Kolmogorov-Smirnov test and the Levene’s test, respectively. Baseline characteristics
and data of the nutrient intake were tested with the independent samples t-tests. A repeated measures ANOVA with the general linear model was used to identify
differences between the two treatments as well as changes over time. For data that
were not normally distributed and/or had heterogeneous variances, a linear mixed model
analysis was applied.

Results

Baseline characteristics and palatability of study products

All 33 individuals randomized in groups AB and BA completed both 8 wk intervention
periods and were included in final analyses (Figure 1). The baseline characteristics of the subjects are shown in Table 2. The consumption of the protein drinks was well accepted by most of the participants
and palatability ratings (evaluation scale from best to worst, 1.0 to 6.0) differed
slightly between MPI (2.2) and LPI drinks (2.7).

Nutrient intake

The analysis of the 5 d food record provided information regarding the composition
of the diet at baseline. Energy and carbohydrate intakes were in accordance with reference
values, whereas protein, fat, and cholesterol intakes were higher compared to recommended
values [19] (Table 3). Nutrient intake following consumption of the 2 d standard diet at wk 8 did not
differ between the two treatments LPI and MPI. In comparison to the diet at baseline,
the intake of energy, protein, and fat was significantly raised during the standard
diet for both protein interventions (P ≤ 0.028).

Table 3.Nutrient intake calculated from the 5 d food record at baseline and from the 2 d standard
diet after 8 wk of intervention with LPI and MPI

Anthropometric data and blood pressure

No treatment effects in anthropometric data or blood pressure were seen at wk 8 among
the two protein interventions (Table 4). Compared to baseline, there was a slight increase in body weight and body fat after
8 wk of intervention with both LPI and MPI (P ≤ 0.045). Systolic blood pressure was significantly reduced after 8 wk of the two
protein interventions (P ≤ 0.014). Diastolic blood pressure and pulse at rest were decreased after 8 wk of
LPI intervention (P ≤ 0.044), whereas they remained constant throughout the MPI intervention.

Table 4.Anthropometric data at baseline and changes after 8 wk of intervention with LPI and
MPI

Plasma parameters

Plasma lipid parameters did not differ between the two treatments, neither at wk 4
nor wk 8, except for a higher HDL cholesterol concentration at wk 4 following intervention
with LPI (P = 0.036, Table 5). Compared to baseline, after 4 wk but not after 8 wk, there was a decrease in LDL
cholesterol following both protein interventions (P ≤ 0.008) and in LDL:HDL cholesterol ratio following LPI intervention (P = 0.003). Concentrations of total cholesterol and triacylglyceroles were not significantly
affected by the protein interventions, except for an increase in triacylglyceroles
after 8 wk of LPI intervention (P = 0.022).

Table 5.Plasma concentrations of blood lipids, hs-CRP, and urea at baseline and changes after
4 wk and 8 wk of intervention with LPI and MPI

Considering the total cholesterol concentrations at baseline, subjects with a higher
initial total cholesterol (> 6.6 mmol/L, n = 14) at an average of 7.6 mmol/L showed
a significant decrease in total and LDL cholesterol after 4 wk of both interventions
compared to baseline (LPI: –0.34 ± 0.59 mmol/L and –0.62 ± 0.52 mmol/L; MPI: –0.47 ± 0.76 mmol/L
and –0.64 ± 0.55 mmol/L; P ≤ 0.048; Figure 2). After 8 wk, a reduction of LDL cholesterol, but not of total cholesterol, was present
following LPI intervention (–0.35 ± 0.54 mmol/L; P = 0.032). LDL:HDL cholesterol ratios were significantly decreased after 4 wk of LPI
and MPI intervention (LPI: –0.68 ± 0.52 mmol/L; MPI: –0.52 ± 0.64 mmol/L; P ≤ 0.009; data not shown). In contrast, in subjects with moderately elevated initial
total cholesterol (≤ 6.6 mmol/L, n = 19) at an average of 5.8 mmol/L, no changes in
total and LDL cholesterol (Figure 2) as well as in LDL:HDL cholesterol ratio (data not shown) could be observed, both
after 4 wk and after either 8 wk of LPI or MPI intervention.

Figure 2.Plasma cholesterol concentrations [mmol/L] at baseline and after 4 wk and 8 wk of
intervention with LPI and MPI in subjects with moderate or higher hypercholesterolemia. The study population was differentiated into two subgroups based on the total cholesterol
concentration at baseline. Subjects with a cholesterol concentration ≤ 6.6 mmol/L
were considered to have moderate hypercholesterolemia (n = 19); subjects with a cholesterol
concentration > 6.6 mmol/L were considered to have higher hypercholesterolemia (n = 14).
LPI, lupin protein isolate; MPI, milk protein isolate. aP-value is for difference between treatments at wk 8 determined by repeated measures
ANOVA. *, *** Significant differences comparing wk 4 and wk 8 with baseline determined by repeated
measures ANOVA (*P ≤ 0.050, ***P ≤ 0.001).

There were no significant differences between the two treatments with respect to hs-CRP
and urea in plasma at wk 4 or wk 8 (Table 5). Concentrations of hs-CRP decreased after 4 wk and 8 wk of LPI as well as MPI intervention
compared to baseline. However, these differences did not reach statistical significance
(P ≤ 0.82). Compared to baseline, the concentrations of plasma urea were increased after
4 wk of both protein interventions (P ≤ 0.001). Following 8 wk of both protein interventions, plasma urea was still higher
compared to baseline (P ≤ 0.022), however, the magnitude was smaller than after 4 wk of intervention.

Serum amino acids

Serum amino acid concentrations at baseline, treatment effects, and changes over time
are shown in Table 6. Except for aspartate, glycine and arginine, the concentrations of proteinogenic
amino acids were significantly higher at wk 4 (P ≤ 0.042) for MPI relative to LPI. However, there were no significant differences
between the two treatments at wk 8. Compared to baseline, the concentrations of almost
all single amino acids increased after 4 wk, but not after 8 wk of intervention with
MPI (P ≤ 0.019). Following the 4 wk and 8 wk intervention with LPI, most of the amino acid
concentrations remained unchanged.

Table 6.Serum concentrations of amino acids at baseline and changes after 4 wk and 8 wk of
intervention with LPI and MPI

Discussion

This randomized crossover study reveals that a modest amount comprising 25.0 g/d of
additionally consumed LPI is capable of lowering total (−5%) and LDL cholesterol concentrations
(–12%) as well as the LDL:HDL cholesterol ratio (−16%) from baseline to wk 4, primarily
in subjects with higher hypercholesterolemia (> 6.6 mmol/L).

The lipid-lowering activity of dietary treatments appears to be strongly dependent
on the subjects’ initial cholesterol concentrations [20,21]. A meta-analysis of 38 human studies on soy protein ascertained that the net changes
in total as well as in LDL cholesterol after intervention were directly related to
the total cholesterol concentration at baseline [20]. A more recent re-evaluation by Sirtori et al. [21] which included a further 33 studies on soy protein confirmed this dependency. In
line with this, a subgroup analysis within the present study revealed that the total
and LDL cholesterol-lowering activities of LPI and MPI were restricted to subjects
with higher initial total cholesterol with an average of 7.6 mmol/L (Figure 2) indicating that there is a similar dependency between cholesterol-lowering activity
and baseline cholesterol concentrations for lupin protein.

As reviewed by Anderson and Konz [22], a 1% increase in either total or LDL cholesterol increases the risk for coronary
heart disease by 2% to 3% and 1%, respectively. Thus, after a 4 wk LPI intervention
the risk for cardiovascular events such as coronary heart diseases would be reduced
by 10% to 15% in subjects with higher hypercholesterolemia. Due to the lack of change
in subjects with moderate hypercholesterolemia, the lipid-lowering effects for the
whole study population were lower. The overall changes in plasma cholesterol after
4 wk of intervention are consistent with two other studies that show a LDL cholesterol-
[8] and a moderate total cholesterol-lowering activity [8,9] of 35.0 g/d lupin protein consumed by hypercholesterolemic subjects over a short-term
period of 4 wk or 6 wk. A recent study conducted by our workgroup in hypercholesterolemic
subjects revealed a decrease in the LDL:HDL cholesterol ratio by 7% after consumption
of 40.0 g/d LPI over 8 wk, whereas total and LDL cholesterol were not altered [12]. In contrast, Belski et al. [23] and Hodgson et al. [24] did not find changes in plasma lipid concentrations in overweight or obese participants
following a long-term intervention from 16 wk to twelve months with an ad libitum diet higher in protein and fiber obtained by enriching foods with lupin flour.

The present study showed a significant reduction in systolic blood pressure by 8.4 mm
Hg after 8 wk of LPI intervention. Since an increase of 1 mm Hg in systolic blood
pressure is expected to increase the risk for coronary heart disease by 2.4% [22] the observed effect in our study could reduce the risk by 20%. These results are
consistent with three previous studies that found a significant decline in blood pressure
following consumption of lupin protein [7] or lupin flour [23,25].

Similar to the findings of Weisse et al. [8], LPI intervention per se only minimally changed amino acid profile (Table 6). The concentrations of methionine were decreased by 8% after 4 wk equivalent to
the observed 7% decline in the study by Weisse et al. [8]. Since LPI relative to MPI had almost a threefold higher arginine and half the lysine
proportion (Table 1), at wk 4, but not at wk 8 of LPI intervention, the lysine:arginine ratio in serum
was significantly lower compared to MPI (Table 6).

Evidently, there was a general decrease in the extent of physiological effects of
both protein interventions from wk 4 to wk 8. This aspect may be explained by a declining
compliance to the study protocol after 4 wk of intervention, which is supported by
a decrease in plasma urea from wk 4 to wk 8 (Table 5). As there was an increase in energy intake (Table 3) as well as in body weight and body fat (Table 4) after 8 wk of both protein interventions compared to baseline, we can presume that
the majority of subjects did not replace an iso-caloric part of their usual diet with the protein drinks. A decrease in body weight
is associated with lower concentrations of triacylglyceroles, total and LDL cholesterol
as well as with higher HDL cholesterol [26]. Thus, the observed weight gain might have additionally contributed to a worsening
of the lipid profile from wk 4 to wk 8.

There were no significant differences in the plasma concentrations of cholesterol
and of hs-CRP or in blood pressure between LPI and MPI intervention. This lack of
treatment effects is not entirely surprising since several studies attribute milk
proteins, particularly several milk peptides, with beneficial physiological properties
such as hypocholesterolemic, hypotensive, and anti-inflammatory activities [3]. Furthermore, increasing evidence indicates that the substitution of protein from
animal as well as plant sources at the expense of carbohydrates may beneficially affect
plasma lipids [4], facilitates loss of body weight [27] and body fat [28], and can lower blood pressure [5]. The mechanisms and bioactive components of lupin protein responsible for the beneficial
effects in the human body have not yet been elucidated [6]. Contrary to soy, proteins from lupin are almost free from isoflavones [29] and thus physiological effects can be attributed to the protein and/or its components
per se. According to Rahman et al. [30], the low lysine:arginine ratio might be responsible for the hypocholesterolemic properties
of lupin protein. As reported by Rajamohan and Kurup [31], a decrease in serum cholesterol in rats was caused by a globulin fraction of sesame
seeds with a low lysine:arginine ratio of 0.67 comparable to the value determined
for the LPI (0.38) used in the present study. However, studies on the impact of different
lysine:arginine ratios on lipid metabolism are lacking. Notably, the high proportion
of arginine amounting to around 10% in lupin protein should be taken into consideration
with regard to the physiological impact. Recent studies indicate that arginine is
capable of modulating the concentrations of lipid signaling molecules [32,33] and the expression of genes involved in the regulation of lipid homeostasis [32] which might lead to changes in the concentration of cholesterol. Hurson et al. [34] investigated the effect of an oral supplementation with 17 g arginine over 2 wk in
elderly subjects. In the arginine-supplemented group, total cholesterol significantly
decreased by 10% due mainly to reduced LDL cholesterol (-10%), whilst HDL cholesterol
remained constant. These observed changes in cholesterol concentrations are in accordance
with the results of the present study after 4 wk of 25 g/d LPI intervention. However,
in the current study, the arginine uptake of 2.5 g/d via LPI was much lower than the supplemented 17 g/d arginine in the study by Hurson et al. [34]. Lupin protein seems to affect the expression of hepatic genes involved in lipid
metabolism as previously shown in hypercholesterolemic rats [35,36] and, further, to alter the activity of LDL receptor as shown in a human hepatoma
cell line [29]. Supporting these results, Weisse et al. [8] observed an increase in mRNA abundance of the sterol regulatory element-binding protein-2
and LDL receptor along with a decrease in mRNA concentrations of 3-hydroxy-3-methylglutaryl-CoA
reductase in mononuclear blood cells from hypercholesterolemic subjects after 6 wk
intervention with 35 g/d lupin protein. Apart from the specific amino acid profile
of lupin protein, several bioactive peptides as well as entire proteins are equally
capable of demonstrating favorable properties [6].

In the present study, we could not detect a triacylglycerole-lowering activity of
LPI. Thus, the inconsistent experimental data referring to the effect of lupin protein
on triacylglyceroles [7-9] indicates the necessity of an inclusion of this parameter in further human studies.
Furthermore, in future studies, it may be desirable to incorporate the test proteins
in usual dietary foods in order to increase the subjects’ compliance and to avoid
changes in dietary composition, thereby sustain body weight and body composition over
the whole study time.

Conclusion

The present study suggests that the supplementation of 25 g/d LPI positively affects
LDL cholesterol and LDL:HDL cholesterol ratio particularly in subjects with higher
hypercholesterolemia. These beneficial effects, were however, largely absent after
8 wk of intervention, due most likely to a declining compliance from wk 4 to wk 8.

Based on our results, we do not expect any adverse effects of lupin protein when integrated
in human nutrition above all because the protein is almost free from isoflavones.
Lupin therefore can be considered as an alternative and valuable source of plant protein
with respect to soy protein. Moreover, supplementation of modest amounts of lupin
protein into the diet could provide a safe and non-pharmacological approach of attenuating
the extent of hypercholesterolemia, thereby reducing the risk of cardiovascular diseases.

Abbreviations

Competing interests

The authors declare that they have no personal or financial conflict of interests.

Authors’ contributions

MB, AF and GJ designed the research; MB was responsible for supervising the study,
sample handling, coordination and conduction of the analyses; MB, JK, and MK analyzed
data; MB performed statistical analysis; MB and GJ were responsible for data interpretation
and had primary responsibility for final content; MB wrote the paper; all authors
read and approved the final manuscript.

Acknowledgements

We thank the Federal Ministry of Education and Research (grant no. 0315683C) for financial
support. Study sponsors were not involved in study design, collection, analysis, and
interpretation of data, in writing the manuscript and in the decision to submit the
manuscript for publication. We thank the Fraunhofer Institute for Process Engineering
and Packaging (Fh-IVV) for supplying protein isolates. Thanks also to the Nutrichem
diät + pharma GmbH for producing the protein drinks. We express thanks to U. Helms
and C. Richert for technical assistance. We are grateful to the biostatistician Dr.
R. Schubert for his assistance in the statistical analysis of data. All authors thank
N. Kroegel for language editing.

References

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